The present invention relates to a biosensor that is manufactured at low cost and can readily determine a specific compound in a sample at high speed and with high accuracy.
Proposed methods of quantitative analysis for determining sugars, such as sucrose and glucose, utilize a technique like polarimetry, colorimetry, reductometry, or chromatography. These methods, however, have relatively low specificity to the sugars and thereby poor accuracy. For example, the polarimetry is simple in operation but significantly affected by the peripheral temperature. Namely the polarimetry is not suitable for domestic use, in which generally non-skilled people determine sugars.
Various types of biosensors recently developed utilize the specific catalytic activities of enzymes.
The following describes determination of glucose as one example of quantitative analysis of a substrate in a sample solution. A known technique of electrochemical determination of glucose uses glucose oxidase (EC1.1.3.4: hereinafter referred to as GOD) and either an oxygen electrode or a hydrogen peroxide electrode (see, for example, `Biosensor` ed. by Shuichi SUZUKI, Kodan-sha).
GOD uses oxygen as an electron acceptor and selectively oxidizes the substrate or .beta.-glucose to D-glucono-.delta.-lactone. In the presence of oxygen, the oxidation reaction by GOD reduces oxygen to hydrogen peroxide. The decrease in amount of oxygen may be measured by the oxygen electrode, or otherwise, the increase in amount of hydrogen peroxide may be measured by the hydrogen peroxide electrode. Both the decrease in amount of oxygen and the increase in amount of hydrogen peroxide are proportional to the content of glucose in the sample solution, so that measurement of the decrease in oxygen or the increase in hydrogen peroxide determines glucose.
As presumable from its reaction process, however, this known technique has some drawbacks: the results of measurement are significantly affected by the concentration of oxygen included in the sample solution; and the absence of oxygen in the sample solution makes the measurement itself impossible.
A novel type of glucose sensor developed to remove such drawbacks uses potassium hexacyanoferrate(III), one of organic compounds such as ferrocene derivatives and quinone derivatives, or a metal complex as the electron acceptor, instead of oxygen. This novel type of sensor oxidizes the reductant of the electron acceptor obtained as a result of the enzyme reaction on the electrode and determines the concentration of glucose included in the sample solution based on the observed oxidation current. Application of the organic compound or metal complex, instead of oxygen, as the electron acceptor enables a known quantity of GOD and the electron acceptor to be accurately carried on the electrode in a stable state to form a reaction layer. The reaction layer that is substantially in a dry state may be integrated with the electrode system. Disposal glucose sensors based on this technique have drawn much attention recently.
This disposal glucose sensor enables the user to readily determine glucose simply by introducing a sample solution into the sensor detachably connected to a measurement device. This technique is applicable to not only determination of glucose but determination of another substrate included in the sample solution.
The technique of utilizing such an electron acceptor and integrating the electrode system with the reaction layer enables simple electrochemical qualitative evaluation of the substrate. Lead conductors composed of a metal, such as palladium or silver, undesirably increase the manufacturing cost. Carbon lead conductors, on the other hand, require a relatively large width to depress an increase in electrical resistance. The large width of lead conductors causes part of a lead conductor led from a working electrode to be exposed to a sample solution, which may result in a positive error in measurement.